Abstract 15544: Implication of the Histone Methyltransferase “G9a” in Pulmonary Arterial Hypertension

Abstract

Rationale: Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder characterized by elevation of pulmonary arterial (PA) pressure and premature death. PA smooth muscle cells (PASMCs) from PAH patients present a cancer-like hyperproliferative and apoptosis-resistant phenotype contributing to remodeling of distal PAs. Although epigenetic alterations contribute to PAH development, one important challenge is defining which genes are the drivers . A growing body of literature points to the role of an epigenetic factor called G9a in cancer pathogenesis. Indeed, G9a is a histone methyltransferase overexpressed in many cancers promoting cell proliferation and survival. Given the similarities between PAH and cancer, it is of interest to determine whether G9a is implicated in PAH. We thus hypothesized that G9a inhibition reduces the pro-proliferative and apoptosis resistance phenotype of PAH-PASMCs. Methods and Results: Using Western blot (WB) and immunofluorescence (IF), we showed that G9a is overexpressed in distal PAs and isolated PASMCs from PAH patients (n= 6-14, p<0.01). Similarly, G9a was increased (WB and IF, p<0.05) in two models mimicking the disease; namely the monocrotaline rat and mice exposed to chronic hypoxia. In vitro, we found that pharmacological inhibition of G9a using BIX01294 and UNC0642 reduces PAH-PASMC proliferation (Ki67 and EdU assays, p<0.001) and survival (Annexin V assay p<0.001). Through RNA sequencing analysis conducted in PAH-PASMCs treated or not with BIX01294, we found that upregulated differentially expressed genes (DEGs) were enriched in cholesterol biosynthesis, autophagy-lysosome and ER stress-induced apoptotic pathways. However, downregulated DEGs were involved in cell cycle and fibrosis-related processes. Consistently, inhibition of G9a generates numerous cytoplasmic vacuoles positive for LC3-II and p62 (WB, IF), thus suggesting that the inhibition of G9a induces cell death by altering cholesterol metabolism-dependent autophagy. Conclusion: We showed for the first time that G9a is overexpressed in PAH contributing to the pro-proliferative and anti-apoptotic phenotype of PAH-PASMCs. Current experiments aim to determine whether G9a inhibition provides therapeutic benefits in PAH.